Enteropathogenic E. coli (EPEC) is an important cause of serious diarrhea in infants in developing countries and a model organism for studies of Enterohemorrhagic E. coli (EHEC) infections. These pathogens are able to attach intimately to host cells and induce dramatic changes in cytoskeletal organization in a process known as attaching and effacing that is dependent on the Locus of Enterocyte Effacement (LEE) pathogenicity island. Ongoing studies of the genes from one end of the LEE have provided important insights into the pathogenesis of EPEC and EHEC infection. Experiments designed to accomplish four specific aims will further these studies. The first aim is to identify the target(s) of and determine the mechanism of action of EspF. Two functions have been identified for EspF. EspF mediates host cell apoptosis and is required for disruption of intestinal barrier function. The mechanism of action of EspF will be revealed by identifying its host cell target(s). These studies will also determine whether these two functions are the result of a single or separate EspF activities. The second aim is to test the hypothesis that EspB has a role in attaching and effacing distinct from its role as a component of the translocation apparatus. EspB is required for translocation of EPEC effector molecules into host cells, but EspB is itself translocated to the host cell cytoplasm and EspB causes dramatic changes in epithelial morphology when transfected and expressed in host cells. Mutants with linker-insertion and point mutations in espB will be screened to determine whether EspB proteins that retain translocation activity but lack attaching and effacing activity can be identified. These studies will also yield a detailed analysis of EspB structure-function relationships. The third specific aim is to test the hypothesis that the product of orf23 is a component of the translocation apparatus and the fourth aim is to test the hypothesis that the product of orf27 is a chaperone for an EPEC effector protein that is not required for attaching and effacing activity. These hypotheses are firmly based on data developed during the current funding period and will lead to new insights into the structure of the translocation apparatus and into functions of the LEE other than attaching and effacing. The experiments described in this proposal are essential for improving our understanding of the pathogenesis of EPEC and EHEC infections.